High-throughput imaging of neuronal activity in Caenorhabditis elegans

Abstract

Neuronal responses to sensory inputs can vary based on genotype, development, experience, or stochastic factors. Existing neuronal recording techniques examine a single animal at a time, limiting understanding of the variability and range of potential responses. To scale up neuronal recordings, we here describe a system for simultaneous wide-field imaging of neuronal calcium activity from at least 20 Caenorhabditis elegans animals under precise microfluidic chemical stimulation. This increased experimental throughput was used to perform a systematic characterization of chemosensory neuron responses to multiple odors, odor concentrations, and temporal patterns, as well as responses to pharmacological manipulation. The system allowed recordings from sensory neurons and interneurons in freely moving animals, whose neuronal responses could be correlated with behavior. Wide-field imaging provides a tool for comprehensive circuit analysis with elevated throughput in C. elegans.M odern neuronal recording techniques are labor-and equipment-intensive, and generally designed to obtain maximal information from individual animals. However, individuals differ from one another. To understand the full range and variability of neuronal responses, it is desirable to apply highthroughput methods and systematic data collection to many animals under controlled stimulation conditions.The nematode Caenorhabditis elegans is particularly amenable to high-throughput studies of neural and behavioral activity, which are facilitated by its small size, compact nervous system, ease of genetic modification, well-defined behavioral repertoire, and transparent body with optical access to single defined neurons. Optical neural recordings in C. elegans have primarily used high-magnification imaging of neurons expressing a genetically encoded calcium indicator such as GCaMP or cameleon. Glued preparations (1) or partial-body traps (2-4) enable imaging of calcium dynamics in head neurons, but immobilization limits the animal's behavioral repertoire. More complex behaviors can be visualized in freely moving animals on agar surfaces by using a moving stage or objective and computer-controlled feedback to track a moving animal and keep a specific neuron in view (5-8). These methods monitor a single animal at a time, as do optical and electrophysiological recording methods in flies, fish, and rodents, which rely upon complex surgeries, implanted sensors, and dedicated equipment for each animal (9-13).Here we describe a strategy for recording neuronal activity evoked by precise chemical stimulation of freely moving or anesthetized C. elegans by using wide-field microscopy. We adapted previous microfluidic arenas optimized for normal C. elegans crawling behavior and repeatable spatiotemporal stimulation (14) to simultaneous optical recording of calcium transients in individual neurons. The automated microscope is capable of continuous recording from more than 20 animals at once for hours during repeated stimulation without user interaction. We demonstrat...

Hierarchical sparse coding in the sensory system of Caenorhabditis elegans

“…2) consists of several stimuli, each tested at a single concentration. It would be interesting to perform an in-depth analysis to quantitate how each of the neurons responds to varying concentrations of the stimuli using dedicated high-throughput microfluidic devices as has been done for amphid wing neuron class A (AWA) (29). Modulating stimulus concentration may somewhat change the ensemble of encoding sensory neurons, often to include the amphid sensilla neuron class H (ASH) polymodal neuron if high concentrations of a known chemoattractant are used (e.g., NaCl and diacetyl) (30,31).…”

Whole-brain calcium imaging with cellular resolution in freely behaving Caenorhabditis elegans

“…24 and and then developing rapidly (17,18,(25)(26)(27)(28)(29). One limitation of these freely moving calcium imaging systems is that they are limited to imaging only a very small subset of neurons and lack the ability to distinguish neurons that lie atop one another in the axial direction of the microscope.…”

An integrated platform enabling optogenetic illumination of Caenorhabditis elegans neurons and muscular force measurement in microstructured environments

“…Enabled by state-of-the-art technologies, a variety of external stimuli has been independently studied as a variable in the worm's environment, including temperature, [15][16][17] chemicals, [18][19][20][21] food-related signals, 22 gas concentration, 23 electric field, 24,25 and physical obstacles. 5,26 When input with these stimuli, the worm was generally treated as a black box to produce observable behaviors.…”

“…[35][36][37] Together with precise position control of agile nematode, optogenetics has been applied to C. elegans to manipulate sensory neurons, interneurons, and motor neurons. 20,[38][39][40][41][42][43][44] Normally, certain neurons of interest in C. elegans can be selectively transferred with a gene that allows the neurons to be switched on/off when projected with light of specific wavelength (or color). In practice, as long as the color light is projected onto the worm body segment that possesses the neurons of interest, the neurons can be stimulated positively or negatively.…”